Screening assays for G protein coupled receptor agonists and antagonists

ABSTRACT

Parathyroid hormone (PTH) and its closely related peptide, PTHrP, share the same receptor, PTHR. LLC-PK1 cells are porcine renal epithelial cells which do not normally express PTHR. The present-invention provides stably transfected LLC-PK1 cells which express human PTHR. Also provided are methods for determining whether a compound of interest is an agonist or antagonist of a Gs or Gq protein coupled receptor.

This Application is a continuation of U.S. application Ser. No.08/903,977, filed on Jul. 31, 1997, which is now U.S. Pat. No.6,183,974, which relied upon and is fully incorporated by referenceherein.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSOREDRESEARCH AND DEVELOPMENT

Part of the work performed during development of this invention utilizedU.S. Government funds. The U.S. Government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to G protein coupled receptors. Morespecifically, screening assays for Gs and Gq protein coupled receptoragonists and antagonists are provided. Also provided are stablytransfected cell lines.

2. Related Art

Parathyroid hormone (PTH) is a major systemic regulator of boneturnover, and the closely related peptide, PTHrP, which is widelyexpressed in adult and fetal tissues, is believed to exert importantparacrine actions, especially in developing cartilage and bone(Dempster, D. W., et al., Endocrine Rev 14:690-709 (1993); Lanske, B.,et al., Science 273:663-666 (1996); Lee, K., et al., Endocrinology137:5109-5118 (1996); Rixon, R. H., et al., J Bone Miner Res 9:1179-1189(1994)). Exogenously administered PTH exerts striking effects upon bonemass in vivo, the nature of which depends critically upon the dose ofPTH and the resulting temporal profile of the concentration ofcirculating peptide (Dempster, D. W., et al., Endocrine Rev 14:690-709(1993)). Thus, continuous exposure to high PTH concentrations leads tonet bone resorption and osteopenia, whereas intermittent administrationof low doses leads to increased net bone formation—a finding that hasfueled great interest in the possible use of PTH, or PTH analogs, asanabolic agents to increase bone mass and to prevent or treat metabolicbone diseases, including osteoporosis (Dempster, D. W., et al.,Endocrine Rev 14:690-709 (1993), whitfield, J. F., and Morley, P.,Trends Pharmacol Sci 16:382-386 (1995)).

Both PTH and PTHrP can activate a single receptor, the PTH/PTHrPreceptor (PTHR), which has been cloned from several species, includingrat, opossum, mouse, pig and human, and shown to be expressed in cellsof bone (Abou-Samra, A. B., et. al., Proc Natl Acad Sci USA 89:2732-2736(1992); Juppner, H., et al., Science 254:1024-1026 (1991); Schneider,H., Eur J Pharmacol 246:149-155 (1993); Bringhurst, F. R., et al.,Endocrinology 132:2090-2098 (1993); Pines, M., et al., Endocrinology135:1713-1716 (1994)). Activation of the PTHR in osteoblasts evokesmultiple parallel signaling events, including activation of adenylylcyclase (AC), phospholipase C (PLC) and cytosolic free calciumtransients (Abou-Samra, A. B., et al., Proc Natl Acad Sci USA89:2732-2736 (1992), Juppner, H., et al., Science 254:1024-1026 (1991),Bringhurst, F. R., et al., Endocrinology 132:2090-2098 (1993), Dunlay,R., and Hruska, K., Am J Physiol 258:F223-231 (1990); Fujimori, A., etal., Endocrinology 128:3032-3039 (1991); Yamaguchi, D. T., et al., JBiol Chem 262:7711-7718 (1987)). The links between each of theseindividual signaling events and the ultimate integrated tissue responsesto PTH, such as changes in overall bone mass, remain largely undefined.It has been reported that PTH analogs which appear to selectivelyactivate AC can generate a full anabolic effect on bone followingintermittent administration in vivo (Rixon, R. H., et al., J Bone MinerRes 9:1179-1189 (1994), Whitfield, J. F., and Morley, P., TrendsPharmacol Sci 16:382-386 (1995), Whitfield, J. F., et al., Calcif TissueInt 58:81-87 (1996)). Such observations have suggested that individualPTH second messengers may indeed be linked to specific tissue responsesand, therefore, that the pattern of PTHR signaling events, as well astheir intensity, may dictate both the qualitative and quantitativeaspects of the response in bone. The issue is complicated by the factthat, in bone, mature osteoclasts are believed not to express PTHRs andthus must experience these influences of PTH only indirectly viaresponses generated by adjacent cells, such as osteoblasts or marrowstromal cells, which do express these receptors (Dempster, D. W., etal., Endocrine Rev 14:690-709 (1993), McSheehy, P., and Chambers, T.,Endocrinology 118:824-828 (1986)). The manner whereby such osteoblasticor stromal PTH target cells might convey, to neighboring cells of theosteoclastic lineage, complex information reflecting subtle differencesin temporal and concentration profiles of PTH exposure remains obscure.

Striking desensitization and downregulation of PTHRs has been describedfollowing exposure to high concentrations of ligand (Fujimori, A, etal., Endocrinology 128:3032-3639 (1991), Abou-Samra, A-B, et al.,Endocrinology 129:2547-2554 (1991); Mitchell, J., and Goltzman, D.,Endocrinology 126:2650-2660 (1990); Fukayama, S., et al., Endocrinology131:1757-1769 (1992)) and it was reported recently that the pattern ofsignaling events generated by the rat PTHR is strongly influenced by thelevel of its expression on the cell surface (Guo, J., et al.,Endocrinology 136:3884-3891 (1995)). Specifically, it was found that themagnitude of the PLC response was directly related to the density ofavailable PTHRs on the surface of stably transfected LLC-PK1 cellsacross a range of expression (40,000-300,000 receptors per cell) thatdid not affect the maximal AC response (Guo, J., et al., Endocrinology136:3884-3891 (1995)).

The human PTHR has been expressed previously in cultured cells (Pines,M., et al., Endocrinology 135:1713-1716 (1994), Schneider, H., et al.,FEBS Lett 351:281-285 (1994), Pines, M., et al., Bone 18:381-389(1996)), but its signaling properties have not yet been elucidatedfully. In particular, the effects of alterations in human PTHRexpression on the character of the signal transduction response(s) havenot been systematically analyzed. Certain amino- or carboxyl-terminallytruncated PTH analogs, such as PTH(3-34), PTH(7-34) and PTH(1-31), havebeen previously found to exhibit selective activation of only a subsetof the usual PTHR second messengers (Rixon, R. H., et al., J Bone MinerRes 9:1179-1189 (1994), Whitfield, J. F., and Morley, P., TrendsPharmacol Sci 16:382-386 (1995), Fujimori, A., et al., Endocrinology128:3032-3039 (1991), Abou-Samra, A. B., et al., Endocrinology135:2588-2594 (1994); Azarani, A., et al., J Biol Chem 271:14931-14936(1996); Chakravarthy, B. R., et al., Biochem Beefiest Res Commum171:1105-1110 (1990); Fujimori, A., et al., Endocrinology 130:29-36(1992); Jouishomme, H., et al., Endocrinology 130:53-60 (1992); Janulis,M., et al., Endocinology 133:713-719 (1993)). The hPTH(1-31) analog wasreported to activate AC but not PKC and yet to retain striking anaboliceffects in ovariectomized rats (Rixon, R. H., et al, J Bone Miner Res9:1179-1189 (1994), Jouishomme, H., et al., J Bone Miner Res 9:943-949(1994)). Confirmation of such selective signaling via human PTHRs wouldprovide important additional rationale for the development of thesesignal-specific PTH peptides for clinical use. Thus, there is a need inthe art for characterization of responses mediated by PTHR.

SUMMARY OF THE INVENTION

The present inventors isolated and characterized numerous subclones ofthe well-characterized renal epithelial LLC-PK1 cell line thatcollectively expressed a broad range of stable transfected human PTHRs.It was found that, as with the rat PTHR, the human receptor activates ACmaximally at levels of receptor expression far lower than those neededfor PLC activation. Further, the temporal pattern and magnitude of PLCactivation in these cells is strongly dependent upon the density ofcell-surface human PTHRs across a range of expression above that whichelicits maximal AC activation. Surprisingly, it was also found thathPTH(1-31) and hPTH(1-34) activate PLC and cytosolic free calciumtransients equivalently via the human PTHR and, moreover, thathPTH(1-31) fully induces other, more delayed biologic responses to PTHin these cells that depend upon cAMP-independent signaling pathways.These findings point to a potential physiologic role of PTHR regulationand differential signaling in fashioning the integrated cellularresponse in tissues such as bone or kidney, and they indicate that theligand selectivity of human PTHRs may differ from that of other speciesof PTHRs.

The present inventors also have developed a convenient and sensitivespectrophotometric bioassay that responds to activation of either AC orPLC via the human PTHR in these cells, and have employed it, togetherwith other measurements, to show that analogs such as PTH(3-34) andPTH(1-31), previously found to be signal-selective agonists in ratcells, may exhibit different spectra of biologic activities via thehuman PTHR.

Thus, the present invention provides a stably transfected cell linecomprising LLC-PK1 cells which express PTHR.

The present invention also provides a method for determining whether acompound of interest is an agonist or antagonist of a Gs or Gq proteincoupled receptor comprising:

-   -   (a) providing a cell line which expresses urokinase-type        plasminogen activator (u-PA);    -   (b) providing an expression vector comprising a nucleotide        sequence encoding for a Gs or Gq protein coupled receptor, said        receptor not normally expressed in said cell line of step (a);    -   (c) introducing said expression vector into said cell line,        thereby providing stably transfected cells;    -   (d) contacting said stably transfected cells with said compound        of interest; and    -   (e) measuring the u-PA activity of the cell culture supernatant        of said cells of step (d).

The present invention further comprises a method of determining whethera compound of interest is an agonist or antagonist of a Gs or Gq coupledreceptor using LLC-PK1 cells in the above-described method. The presentinvention further provides a method of determining whether a compound ofinterest is an agonist or antagonist of human PTHR using theabove-described method.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B and 1C-1D. Competitive radioligand binding to humanPTH/PTHrp receptors stably expressed in LLC-PK1 cells. LLC-PK1 cellsstably expressing (FIGS. 1A-1B) 950,000 receptors/cell (HKRK B7) or(FIGS. 1C-1D) 280,000 receptors/cell (HKRK B28) were incubated with¹²⁵I-[Nle^(8,18,)Tyr³⁴]bPTH(1-34) in binding buffer for 6 hr at 15° C.in the presence or absence of increasing concentrations ofnonradioactive ligand. Scatchard plots are shown in the insets. Eachpoint represents the mean± of triplicate determinations. Totalradioligand bound ranged from 15,000-30,000 cpm/well, and nonspecificbinding was 2-5% of total binding.

FIGS. 2A-2B. Stimulation of cAMP accumulation in LLC-PK1 cellsexpressing human PTH/PTHrP receptors. Cyclic AMP was measured in acidextracts of cells prepared after addition of the indicated peptides andincubation at 37° C. for 20 min. FIG. 2A shows the responses tohPTH(1-34), at the indicated concentrations, in HKRK B7 cells (●;950,000 receptors per cell) and HKRK B28 cells (∘; 280,000 receptors percell). FIG. 2B shows the responses in HKRK B28 cells to: hPTH(1-34) (∘),hPTH(1-31) (Δ), hPTH(3-34) (▪), hPTH(7-34) (□), hPTHrp(1-36) (♦), andsCT (Δ). Results were expressed as fold basal. Each point represents themean±SEM of triplicate determinations.

FIG. 3. Influence of PTH/PTHrP receptor density upon maximal activationof PLC by PTH in LLC-PK1 cells expressing human receptors. The maximalincrease in IP₃ production, expressed as percentage of basal, is shownfor LLC-PK1 cells expressing different densities of human (▪) and rat(□) PTH/PTHrp receptors. Cells were stimulated for 30 min withhPTH(1-34) at a concentration of 1000 nM, which elicits maximalactivation of this response (see FIG. 4B). Each point depicts themean±SEM of three experiments performed in triplicate. The relationbetween PLC activity and receptor expression is shown schematically bythe dashed lines for the rat (left) and human (right) receptors.

FIGS. 4A-4B. Comparison of time-and dose dependence of IP₃ productionbetween HKRK B7 and HKRK B28 cells. FIG. 4A shoes HKRK B7 cells (●) andHKRK B28 cells (∘) that were stimulated with hPTH(1-34) (1000 nM) forthe indicated time. FIG. 4B shows cells that were stimulated withhPTH(1-34) at the indicated concentrations for 30 min (HKRK B7) or 4 min(HKRK B28). Results are expressed as percent of basal, and each point isthe mean±SEM of three experiments performed in triplicate.

FIGS. 5A-5D. Cytosolic free calcium responses to hPTH in LLC-PK1 cellsexpressing human PTH/PTHrp receptors. Cytosolic free calcium wasmeasured in fura 2AM-loaded confluent monolayers of cells. Responseswere measured in HKRK B7 cells following addition of hPTH(1-34) (FIG.5A) or hPTH(1-31) (FIG. 5B); in HKRK B28 cells following hPTH(1-34)(FIG. 5C); and in AB45 cells following hPTH(1-34) (FIG. 5D). Basalcytosolic free calcium concentrations and those following addition ofsCT in these cell lines were virtually identical—i.e. 20-50 nM and400-500 nM, respectively. These results shown are representative of atleast 5 independent experiments with each cell line.

FIGS. 6A-6B. Stimulation of urokinase-type plasminogen activator (u-PA)secretion by LLC-PK1 cells . Urokinase-type plasminogen activatoractivity was measured in conditioned medium of (FIG. 6A) HKRK B7 cellsor (FIG. 6B) HKRK B28 cells, 16 hr after addition of agonists asfollows: HPTH(1-34) (concentration shown in nM), 8BrcAMP (1 mM), TPA(100 nM), or sCT (1000 nM). Results are expressed in Ploug units/wellusing purified human urokinase as a standard. Each bar depicts themean±SEM of a representative experiment performed in triplicate. Similarresults were obtained in over 10 individual experiments.

FIG. 7. Stimulation of u-PA activity in LLC-PK1 cells that expressdominant-negative inhibition of protein kinase A. Secretion of u-PA wasmeasured 16 hr after addition of agonist to AB45 cells, which co-expresshuman PTHRs and REV AB, a dominant-negative inhibitor of PKA. Resultsare expressed in Ploug units/well, using purified human urokinase as astandard. Each point depicts the mean±SEM of a representative experimentperformed in triplicate. Similar results were obtained from at least 5individual experiments.

FIG. 8. Regulation of phosphate uptake by PTH in HKRK B7 and HKRK B28cells. Phosphate uptake was measured in HKRK B7 cells (closed bars) andHKRK B28 cells (open bars) following incubation for 6 hr in serum-freemedium containing vehicle, hPTH/PTHrp peptides (1000 nM), 8BrcAMP (1mM), TPA (100 nM), or sCT (1000 nM). Preliminary experiments (not shown)demonstrated maximal responses to hPTH/PTHrp peptides at concentrationsof 1000 nM or above. Results are expressed as percentage of basal, andeach bar represents the mean±SEM of a representative experimentperformed in triplicate. Similar results were obtained from at least 5individual experiments.

FIG. 9. Stimulation of u-PA secretion by hPTH(1-31) and hPTH(1-34) inHKRK B7 cells. Urokinase-type PA activity was measured 16 hr afteraddition of hPTH(1-34) (●) or hPTH(1-31) (Δ) at the indicatedconcentrations and expressed as Ploug units/well using purified humanurokinase as a standard. Each point is the mean±SEM of a representativeexperiment performed in triplicate. Similar results were obtained in atleast 6 individual experiments.

FIG. 10. Stimulation of cyclic AMP accumulation by hPTH(1-31) andhPTH(1-34) in LLC-PK1 cells that express rat PTHRs. Cyclic AMPaccumulation was measured in response to hPTH(1-34) (●) or hPTH(1-31)(Δ) in EW5 cells that expressed 320,000 rat PTHRs per cell. Results areexpressed as fold basal.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present inventors have developed LLC-PK1 cell lines which expresshuman PTHR The present studies of clonal LLC-PK1 cell lines thatcollectively span a broad range of expression of stably transfectedhuman PTHRs have demonstrated receptor density-dependent differences inhPTH signaling and biologic activity that indicate an important role forregulation of human receptor expression in modulating the character, aswell as the magnitude, of the cellular response to the hormone.

In one embodiment of the present invention, a stably transfected cellline is provided comprising LLC-PK1 cells which express human PTHR. Acell line is a population of cells of the same type that is capable ofindefinite survival in culture. By “stably transfected cell line” it ismeant that the cell line has been altered in some way to express apolypeptide which it does not normally express. By “express” it is meantthat a structural gene is transcribed into mRNA and that such rnRNA istranslated to produce a polypeptide. LLC-PK1 cells are porcine renalepithelial cells which express calcitonin and vasopressin receptors, butdo not normally express PTHR and are available from the American TypeCulture Collection, ATCC No. CL-101. The human PTHR is a receptor whichbinds to both human PTH and human PTHrP. The human PTHR has beenpreviously cloned (Schneider et al., Eur. J. Pharmacol. 246:149-155(1993); Adams et al., Biochem. 34:10553-10559 (1995)). Thus, LLC-PK1cells which express human PTHR are said to be “stably transfected.”

It has previously been shown that u-PA is secreted by LLC-PK1 cells inresponse to activation of both the PKA and PKC pathways by calcitonin(Jans, D. A., and Hemmings, B. A., FEBS Lett 205:127-131 (1986)). Thepresent inventors have reconfirmed that, in LLC-PK1 cells, both the PKAand PKC pathways are linked to u-PA production. The present inventorshave developed a spectrophotometric bioassay that measures u-PAproduction, and thus PKA or PKC activation, in these cells. An agonistof a Gs or Gq protein coupled receptor increases u-PA production incells which express u-PA. An antagonist of a Gs or Gq protein coupledreceptor inhibits the activity of a Gs or Gq protein coupled receptoragonist, thereby decreasing u-PA production in cells which express u-PArelative to the agonist administered alone.

Cell lines other than LLC-PK1 express u-PA. These cell lines could beused to determine whether a compound is an agonist or antagonist of a Gsor Gq protein coupled receptor using the method of the presentinvention. Examples of cells lines which express u-PA include, but arenot limited to, HE-LU (Rifkin) (ATCC No. CRL-7717); LLC-MK2 (ATCC No.CCL-7); NMU (ATCC No. CRL 1743); LLC-RK1 (ATCC No. CCL-106); and MIAPaCa-2 (CRL-1420).

Thus, a further embodiment of the present invention involves a method ofdetermining whether a compound of interest is an agonist or antagonistof a Gs or Gq protein coupled receptor comprising: (a) providing a cellline which expresses urokinase-type plasminogen activator (u-PA); (b)providing an expression vector comprising a nucleotide sequence encodingfor a Gs or Gq protein coupled receptor, said receptor not normallyexpressed in said cell line of step (a);(c) introducing said expressionvector into said cell line, thereby providing stably transfected cells;(d) contacting said stably transfected cells with said compound ofinterest; and (e) measuring the u-PA activity of the cell culturesupernatant of said cells of step (d). A preferred embodiment of thepresent invention is a method of determining whether a compound ofinterest is an agonist or antagonist of a Gs protein coupled receptor,using the above-described method. Another preferred embodiment of thepresent invention is a method of determining whether a compound ofinterest is an agonist or antagonist of a Gq protein coupled receptor,using the above-described method. An especially preferred embodiment ofthe present invention is a method of determining whether a compound ofinterest is a Gs or Gq protein coupled receptor agonist or antagonistusing LLC-PK1 cells in the above-described method. Another especiallypreferred embodiment of the present invention is a method of determiningwhether a compound of interest is an agonist or antagonist of human PTHRusing the above-described method.

A “compound of interest” could be a peptide, a polypeptide, a fragmentof a polypeptide, an organic natural molecule, or a synthetic molecule.Examples of compounds that could be agonists or antagonists of Gs or Gqprotein coupled receptors are hormones, hormone analogs, and antibodies.

An “agonist of a Gs or Gq protein coupled receptor” is a compound thatinteracts with a Gs or Gq protein coupled receptor and activates the Gsor Gq protein coupled receptor. An “antagonist of a Gs or Gq proteincoupled receptor” is a compound that inhibits the agonist-inducedactivation of a Gs or Gq protein coupled receptor.

A “Gs or Gq protein coupled receptor” is a receptor that, when bound toits appropriate ligand, activates a Gs or Gq protein. Some receptors canactivate both Gs and Gq proteins, while some activate only either Gs orGq proteins. Preferably, the Gs or Gq protein coupled receptors used inthe present invention should be capable of activating Gs or Gq proteinsof the cells used in the method of the present invention. Examples of Gsprotein coupled receptors include, but are not limited to, theβ-adrenergic, glucagon, ADH, FSH, LH, and VIP receptors. Examples of Gqprotein coupled receptors include, but are not limited to, the TRH,thrombin, and PGF_(2α) receptors. One example of a receptor whichactivates both Gs and Gq proteins is the calcitonin receptor.

As used herein, an “expression vector” is a vector comprising astructural gene operably linked to an expression control sequence sothat the structural gene can be expressed when the expression vector isstably transfected into an appropriate host cell. Two DNA sequences aresaid to be “operably linked” if the nature of the linkage between thetwo nucleic acid molecules does not (1) result in the introduction of aframe-shift mutation, (2) interfere with the ability of the promoterregion sequence to direct the transcription of the desired sequence, or(3) interfere with the ability of the desired sequence to be transcribedby the promoter region sequence. Thus, a promoter region would beoperably linked to a desired nucleic acid sequence if the promoter werecapable of effecting transcription of that nucleic acid sequence.Preferred promoters include the promoter of the mouse metallotionein Igene (Harner, D. et al., J. Mol. Appl. Gen. 1:273-288 (1982)), the HSVthymidine kinase promoter (McKnight, S. Cell 31:355-365 (1982)) or theSV40 early promoter (Benoist, C. et al., Nature 290:304-310 (1981)).

Expression of Gs or Gq protein coupled receptors in cells may beincreased by inserting an enhancer sequence into the expression vector.Enhancers are cis-acting elements of DNA, generally about 10 to 300 bpin size, that act to increase transcriptional activity of a promoter.Illustrative examples of enhancers include, but are not limited to, theSV40 enhancer, which is located on the late side of the replicationorigin at bp 100 to 270; the cytomegalovirus early promoter enhancer;the polyoma enhancer on the late side of the replication origin; andadenovirus enhancers.

The expression vectors may provide for inducible expression of thenucleic acid sequence. Preferred among such vectors are vectors whichprovide for expression that is inducible by environmental factors thatare easy to manipulate, such as temperature and nutrient additives.

The expression vectors may also contain a selectable marker forpropagation in stably transfected cells. Representative examples ofselectable markers include dihydrofolate reductase, hygromycin orneomycin resistance.

An expression vector can be provided commercially or it can beconstructed through any method of cloning well-known in the art.Preferred expression vectors include, but are not limited to, pWLNEO,pSV2CAT, pOG44, pXT1, and pSG available from Stratagene; psVK3, pBPV,pMSG and pSVL available from Pharmacia; and pcDNAIneo available fromInvitrogen. Other suitable vectors will be readily apparent to theskilled artisan.

The expression vector can be introduced into the host cell by anyappropriate method, including infection, transduction, transfection,transvection, electroporation, and transformation. Such methods aredescribed in many standard laboratory manuals, such as Davis et al.,Basic Methods in Molecular Biology (1986).

By “not normally expressed in the cell line” it is meant that the Gs orGq protein coupled receptor is not expressed at a detectable level inthe cell line used in the method of the present invention.

By “contacting stably transfected cells with a compound of interest” itis meant that the compound of interest is administered to the stablytransfected cells by any appropriate method. This can includeadministering the compound exogenously in vitro or in vivo, or furthertransfecting the cells to express the compound. When administering thecompound exogenously, a carrier may be used. The carrier should becompatible with cell viability. Examples of carriers include, but arenot limited to, DMSO, MeOH/EtOH, water, Tris, and HEPES.

The compound of interest may be highly purified, partially purified, orunpurified. Compounds of interest may be administered to the stablytransfected cells alone or in combination. By administering thecompounds in combination, synergistic effects on the Gs or Gq proteincoupled receptor may be determined.

By “measuring the u-PA activity” it is intended qualitatively orquantitatively measuring or estimating the level of u-PA activity eitherdirectly (i.e., by determining orestimating absolute u-PA activity) orrelatively (i.e., by comparing the u-PA activity of the stablytransfected cell line that has been contacted with a compound ofinterest to a control stably transfected cell line that has not beencontacted with the compound of interest). Preferably, the u-PA activityin the stably transfected cell line that has been contacted with acompound of interest will be compared to the u-PA activity of either thestably transfected cell line which has not been contacted with thecompound of interest or an untransfected cell line which has beencontacted with the same compound of interest, or both.

One preferred method of measuring u-PA activity is described in Example3 below. Briefly, an aliquot of supernatant from stably transfectedcells is transferred to a clean microplate. A “supernatant” is theliquid medium which has been removed from the cells. A buffer forassaying u-PA activity is added. The microplate is incubated for anappropriate amount of time, and the reactions are stopped with atermination buffer. Absorbance of the colorimetric product is measured.

Preferably, when determining whether a compound of interest is anagonist, the compound is administered to stably transfected cells andlevel of u-PA activity is measured or estimated and compared to thelevel of u-PA activity in stably transfected cells which have not comein contact with the compound of interest.

When determining whether a compound of interest is an antagonist, boththe compound of interest and a known agonist should be administered tothe stably transfected cells. The agonist and potential antagonist canbe administered in any order (ie., agonist first and then potentialantagonist or potential antagonist first and then agonist), as well asconcurrently. After contacting the cells with both an agonist and thecompound of interest, the level of u-PA activity in the stablytransfected cells is measured or estimated and compared to the level ofu-PA activity in stably transfected cells which have been contacted withthe agonist alone.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration only, and are not intended to be limiting of thepresent invention.

EXAMPLE 1 Plasmid Transfection and Isolation of Clones

The cloned porcine kidney-derived cell line, LLC-PK1 (Bringhurst, F. R.,et al., Endocrinology 132:2090-2098 (1993)) and subclones isolated afterstable transfection with human PTH/PTHrp receptor cDNA were maintainedin DMEM supplemented with 7% FBS and 1% penicillin/streptomycin with orwithout 1000 ug/ml G418 (all from GIBCO-BRL, Grand Island, N.Y.) under5% CO₂ in air. To prepare cell lines stably expressing the humanPTH/PTHrP receptor, confluent monolayers of cells were transfected witha full-length human PTH/PTHrP receptor cDNA constructed in the mammalianexpression vector pcDNAIneo (Invitrogen, San Diego, Calif.) (Schipani,E., et al., Science 268:98-100 (1995)). In some cases, cells wereco-transfected with the receptor cDNA and a plasmid encoding adominant-negative PKA regulatory subunit mutant, “REV AB” (Clegg, C. H.,et al., J Biol Chem 262:13111-13119 (1987)). Transfections wereperformed using the calcium-phosphate precipitation technique aspreviously described (Bringhurst, F. R., et al., Endocrinology132:2090-2098 (1993)).

Eleven clonal cell lines were selected from among a total of over 100independent subclones to provide a broad range of receptor expression(Table 1).

Table 1. Human PTH/PTHrP Receptor Expression and Signaling in ClonalLLC-PK1 Cells

Subclones of LLC-PK1 cells stably transfected with hPTHR cDNA wereanalyzed by competitive radioligand (Scatchard) binding analysis and formaximal signaling responses (mean±SD of fold- or percent of basal)) tohPTH(1-34) (1000 nM). All values were obtained from at least two orthree experiments performed in triplicate. Cyclic AMP accumulation andIP₃ formation in controls ranged from 3 to 10 pmol/well and from 1,000to 1,500 cpm/well, respectively.

TABLE 1 Receptor Apparent IP₃ Number/Cell Kd cAMP Accu- FormationSubclone (×10³) (nM) mulation (% of Basal) Human Receptor HKRK B64 901.3 13 ± 2 116 ± 18 HKRK C53 120 1.2  58 ± 16 112 ± 4  HKRK B28 280 1.8103 ± 17 107 ± 1  HKRK C101 330 1.6 94 ± 1 114 ± 11 HKRK C27 340 2.2 64± 8 113 ± 10 HKRK C30 600 2.8 — 192 ± 44 HKRK B5 840 3.9 — 462 ± 47 HKRKB57 900 5.6 — 243 ± 31 HKRK B7 950 4.5  99 ± 10 298 ± 21 HKRK B54 10206.4 — 287 ± 19 HKRH C58 1030 3.6 —  642 ± 106 Rat Receptor EW29 190 2.5148 ± 16 289 ± 16 EW5 320 2.3 130 ± 6  480 ± 14

EXAMPLE 2 Radioligand Binding

Specific binding to the PTH/PTHrp receptor was measured as previouslydescribed (Bringhurst, F. R., et al., Endocrinology 132:2090-2098(1993)), with some modification. In brief, the cells were seeded into24-well plates at a density of 2.5×10⁵ cells/well and cultured for afurther 2 days before study. Cell layers were rinsed with ice-coldBuffer A [50 mM Tris-HCl (pH 7.7), 100 mM NaCl, 2 mM CaCl₂, 5 mM KCl,0.5% FBS, and 5% heat-inactivated horse serum] and then incubated with¹²⁵I-labeled [Nle^(8,18), Tyr³⁴]bovine (b) PTH(1-34) (1×10⁵ cpm/well),with or without unlabeled peptide, in 0.5 ml of Buffer A for 6 h in thecold room (2-8° C.). The binding reaction was terminated by aspiratingthe incubation mixture, after which the cells were washed twice with 0.5ml of ice-cold Buffer A. After solubilizing the cells with 0.5 ml ofLysis Buffer (0.5 N NaOH+0.1% Triton X-100), measurements ofradioactivity and protein were performed to calculate the receptornumber per cell by Scatchard analysis, as previously described(Bringhurst, F. R., et al., Endocrinology 132:2090-2098 (1993)). Allreagents, unless otherwise specified, were obtained from Sigma (St.Louis, Mo.), and all isotopes were purchased from Dupont-New EnglandNuclear (Boston, Mass.). The [Nle^(8,18), Tyr³⁴]bPTH(1-34) wasradioiodinated by the chloramine-T method and purified as previouslydescribed (Bringhurst, F. R., et al., Endocrinology 132:2090-2098(1993)).

Radioligand competition assays and Scatchard analyses for tworepresentative cell lines, HKRK B7 and HKRK B28, which express 950,000and 280,000 hPTHRs per cell, respectively, are shown in FIGS. 1A-1D.Scatchard analysis of the all of the selected cell lines demonstrated arange of PTHR expression from 90,000 to 1,000,000 sites per cell, withapparent K_(d)'s between 1 and 7 nM (Table 1). In each case, theScatchard analysis was linear, consistent with a single class of highaffinity binding sites. The subsequent example described below wasconducted with two cell lines, which were chosen as representative ofthose expressing relatively high (HKRK B7) and low (HKRK B28) densitiesof hPTHRs, respectively.

EXAMPLE 3 Human PHTR Signaling in LLC-PK1 Cells

Methods

Peptides and Other Reagents

All reagents, unless otherwise specified, were obtained from Sigma (St.Louis, Mo.), and all isotopes were purchased from Dupont-New EnglandNuclear (Boston, Mass.). [Nle^(8,18), Tyr³⁴]bPTH(1-34),[Tyr³⁴]hPTH(1-34), hPTH(1-31), hPTH(3-34), hPTH(7-34), and hPTHrp(1-36)were synthesized with carboxy-terminal arnide groups. The [Nle^(8,18),Tyr³⁴]bPTH(1-34) was radioiodinated by the chloramine-T method andpurified as previously described (Bringhurst, F. R., et al.,Endocrinology 132:2090-2098 (1993)).

Cellular cAMP Accumulation

Cells were seeded into 24-well plates at a density of 2.5×10⁵ cells/welland cultured for a further 2 days before study. The cells were rinsedonce with 0.5 ml of ice-cold Buffer B [10 mM HEPES (pH 7.4), 130 mMNaCl, 5 mM KCl, 1.2 mM CaCl₂, 1 mM MgCl₂, 1.2 mM Na₂HPO⁴ 5 mM glucose,and 0.1% heat-inactivated BSA] supplemented with 1 mMisobtylmethylxanthine (IBMX), and placed on ice. Treatments were addedto each well in 0.25 ml of IBMX-supplemented Buffer B, after which theplates were incubated at 37° C. for 15 min. The buffer then was rapidlyaspirated, the plates were rapidly transferred onto liquid nitrogen, andthe frozen monolayers were subsequently thawed directly into 0.5 ml of50 mM HCl. The extracted cAMP then was measured using a commercial RIAkit (Dupont-New England Nuclear, Boston, Mass.). Results were expressedas fold-basal, where basal levels ranged between 38 and 128 pmol/mgprotein/15 min. The basal level of cAMP accumulation in native LLC-PK1cells was not consistently altered in subclones that expressed wild typePTH/PTHrp receptors.

Inositol 1,4,5-triphosphate (IP₃) Production

Cells were seeded into 24-well plates at a density of 2.5×10⁵ cells/welland cultured for a further 2 days before study. The cells were labeledat 37° C. for 16 h before assay with 3 uCi/ml of [³H]myo-inositol inassay medium (serum- and inositol-free DMEM (GIBCO-BRL, Grand Island,N.Y.) supplemented with 0.1% BSA). After washing the cells withprewarmed assay medium containing 30 mM LiCl, they were incubated with0.25 ml of assay medium containing 30 mM LiCl and stimulators (orvehicle) at 37° C. for various intervals(1-30 min). IP₃ formation wasarrested by aspiration and immediate addition of 0.5 ml of ice-cold 5%TCA. The acid extracts then were extracted twice with two volumes ofwater-saturated ether and adjusted to pH 7.4 with concentrated NaOH andTris base (final concentration=10 mM) prior to chromatography on AG 1×8anion-exchange columns (Bio-Rad, Richmond, Calif.), as previouslydescribed (Guo, J., et al., Endocrinology 136:3884-3891 (1995)). Aftereluting other fractions (free [³H]myo-inositol, glycerophosphateinositol, IP₁, and IP₂), IP₃ fractions were collected and their contentof radioactivity was determined by liquid scintillation spectrometry(Beckman, model LS 6000IC).

Measurement of Cytosolic Free Calcium

Cytosolic free calcium was measured by dual fluorescence in cells loadedwith the Ca²⁺-sensitive intracellular probe fura-2. Cells were seededonto glass coverslips at a density of 40,000-600,000/cm² and incubatedfor 2 days as described above. Coverslips were washed twice with PBSbefore loading in phosphate-free Buffer B containing 4 uM of fura-2/AM(Molecular Probes, Eugene, Oreg.) for 30 min at room temperature (22°C.). After washing twice with fura-free Buffer B, the coverslips weremounted in a cuvette containing 2 ml of phosphate-free Buffer B at 37°C. and the emission at 510 nm in response to alternating excitation at340 and 380 nm was monitored using a ratiometric PTI Deltascanfluorescence spectrometer with excitation and emission bandwidths of 2nm. After achieving a stable baseline, agonists were introduced byexchange into 2 ml of fresh buffer to which 10-20 ul of concentratedagonist stock (or vehicle alone) had been added. Peptide stocks wereprepared in 0.1% trifluoroacetic acid, ionomycin and phorbol esters weredissolved in dimethyl sulfoxide and all other additives were prepared inwater. Maximum and minimum fluorescence signals were obtained byexposure to modified buffers containing 1 uM ionomycin plus 20 mMcalcium or 2 mM EGTA in the nominal absence of calcium, respectively,and autofluorescence was estimated from signals obtained in the presenceof ionomycin plus 2.5 mM MnCl₂. Cytosolic free calcium then wasdetermined as previously described (Bringhurst, F. R., et al.,Endocrinology 132:2090-2098 (1993)).

Urokinase-type Plasminogen Activator (u-PA)

Cells were seeded into 96-well plates at a density of 6×10⁴ cells/welland used the following day. The cells were washed once with 0.2 ml andthen refed with 0.1 ml of prewarmed DMEM containing 0.05% BSA. Afteradding each stimulator, plates were returned to the incubator at 37° C.for 16 h. Conditioned medium (5 ul) then was transferred from each wellto a clean microplate. Reactions were initiated by addition of 50 ul ofu-PA assay buffer [90 mM Tris-HCl (pH 8.8), 0.45% Triton X-100, 14 ug/mlhuman plasminogen (Calbiochem, San Diego, Calif.) and 0.4 mg/ml S-2251plasmin substrate (D-Val-Leu-L-Lys-NH-Np) (Sigma, St. Louis, Mo.)].Plates were incubated first at 37° C. for 15 min and then at roomtemperature (22° C.) for 30 min. The reactions were terminated byaddition of 10 ul of ice-cold 25% acetic acid. Absorbance ofcalorimetric product was measured at 405 nm within 1 hour. Activity wasexpressed in Ploug units/well, using purified human urokinase(Calbiochem) as a standard.

Cellular Phosphate Uptake

Cells were seeded into 24-well plates at a density of 2.5×10⁵ cells/welland cultured for a further 2 days before study. Twenty four hours beforeassay, cells were refed with 0.5 ml of DMEM containing 0.1% BSA. Thenext day, cells were refed with serum-free medium (DMEM+0.1% BSA)containing drugs or peptides, and the incubations were continued at 37°C. under 5% CO₂ in air for another 6 hr. Cells then were washed withprewarmed Buffer B without BSA and Na₂HPO₄ and then were incubated at37° C. for 5 min in 0.25 ml of the same buffer containing[³²P]orthophosphate (4 uCi/ml) and 0.1 mM Na₂HPO₄. Phosphate uptake wasarrested by adding 1 ml of ice-cold sodium-free “stop buffer” [10 mMHEPES (pH 7.4), 140 mM choline chloride, and 5 mM sodium arsenate],followed by two further washes with same buffer. The drained cell layerthen was solubilized in 0.5 ml of Lysis Buffer, and cell-associatedradioactivity was determined by Cerenkov counting, as previouslydescribed (Guo, J., et al., Endocrinology 136:3884-3891 (1995)).

Results

The investigations of the properties of human PTHRs, stably expressed inLLC-PK1 cells, have produced two important observations. The first isthe finding that the cell-surface density of the human PTHR is a keydeterninant, independent of ligand concentration, of the magnitudes ofthe individual signaling responses to PTH and that these changes occurover quite different, essentially non-overlapping, ranges of receptordensity. In the course of these studies, hPTHR density-dependentdifferences in the temporal profile of the PLC response were alsoobserved, and evidence of a role for PKA in sustaining the initialcytosolic calcium signal elicited by PTH in these cells was detected.The second major finding is that the putative signal-selective PTHanalog hPTH(1-31) is not at all signal-selective in cells bearing humanPTHRs, even though its PLC activating capacity is somewhat reduced incells that express the rat PTHR. These results point to subtledifferences in ligand-receptor interaction between human and otherspecies of PTHRs and suggest that conclusions derived from rat modelsshould be extrapolated with caution to human PTHR-expressing systems.

Influence of Cell-surface Density Upon Signaling Properties of ExpressedhPTHRs

Maximal PTH-dependent cAMP accumulation was assessed in the humanPTHR-transfected LLC-PK1 subclones following incubation for 15 min witha high concentration (1000 nM) of hPTH(1-34) (Table 1). As reportedpreviously for LLC-PK1 cells that stably expressed rat PTHRs (Guo, J.,et al., Endocrinology 136:3884-3891 (1995)), the maximal adenylylcyclase response was relatively independent of the surface density ofhuman PTHRs above a relatively low threshold (relative to that requiredfor PLC activation—see below). In the case of the human PTHR, however,this threshold for saturation of maximal AC activation appeared to liebetween 120,000 and 280,000 (Table 1), which is at least 6 to 10-foldhigher than that previously observed for the rat PTHR (Guo, J., et al.,Endocrinology 136:3884-3891 (1995)). As expected, activation of AC byhPTH(1-34) in hPTHR-expressing LLC-PK1 cells was concentration-dependentand, as shown in FIG. 2A, the EC50's (approximately 1 nM) and maximalresponses were identical in the cell lines HKRK B28 and HKRK B7.

In previous studies involving expression of rat PTHRs in LLC-PK1 cells,maximal PLC activation by PTH, measured as total IP₃ released at 30 min,was found to be closely related to the density of rat PTHRs across awide range of expression (i.e. from 20,000 to 400,000 receptors percell) that clearly exceeded the threshold of receptor density requiredfor saturation of maximal AC activation (Guo, J., et al., Endocrinology136:3884-3891 (1995)). Similarly, as shown in FIG. 3 and Table 1,maximal PLC activation was strongly dependent upon PTHR density amongLLC-PK1 subclones expressing human PTHRs. As in the case of ACactivation, the relative efficiency with which the human PTHR coupled toPLC in these cells was approximately 3-5-fold less than that of the ratreceptor.—i.e., roughly 3-5-fold more human than rat receptors wererequired for equivalent activation of PLC.

In several PTH-responsive cell lines that express lower numbers ofPTHRs—i.e. UMR 106 cells (approximately 70,000 receptors per cell)—apattern of very rapid but transient PLC activation has been described(Yamaguchi, D. T., et al., J Biol Chem 262:7711-7718 (1987)). Seeking todetermine if such a transient pattern might underlie the absence of asustained PLC activation in LLC-PK1 cells expressing fewer than 400,000human PThRs, preliminary studies were performed that demonstratedincreased IP₃ at an earlier time (4 min) in HKRK C53, HKRK B28 and HKRKC101 cells (120,000, 280,000 and 330,000 PTHRs per cell, respectively),though not in HKRK B64 cells (90,000 sites per cell) (data not shown).The time course of PLC activation then was studied in more detail inHKRK B28 cells and compared with that in HKRK B7 cells. As shown in FIG.4A, transient PTH-stimulated IP₃ formation was observed in HKRK B28cells within the first few minutes of PTH exposure. This response peakedat 4 min and was followed by a rapid decrease to basal levels by 10 min.In contrast, as previously reported for LLC-PK1 cells that expressed300,000 rat PTHRs/cell (Guo, J., et al., Endocrinology 136:3884-3891(1995)), IP₃ formation in HKRK B7 cells continued to increase between 10and 30 minutes, although a more rapid initial phase also was evident inthese cells.

The different temporal patterns of PTH-induced PLC activation observedin HKRK B28 and HKRK B7 cells suggested several possibilities, includingmore extensive desensitization, which might more rapidly extinguish theweaker PLC response in HKRK B28 cells, or possibly recruitment, viahigher receptor expression, of a more weakly coupled but distinctmechanism of IP₃ formation in the HKRK B7 cells. In an effort todistinguish these, it was assessed whether, as reported in other systems(Menniti, F., et al., Mol Pharmacol 40:727-733 (1991)), theconcentration dependence might be different for the rapid (i.e. 4 min)vs. the sustained (i.e. 30 min) PLC responses in HKRK B28 and HKRK B7cells, respectively. As shown in FIG. 4B, however, although the extentof the maximal responses at 4 min in HKRK B28 and at 30 min in HKRK B7were very different, the EC₅₀'s of the two responses were identical(approximately 20-30 nM) and similar to that previously reported for therat PTHR in these cells (Guo, J., et al., Endocrinology 136:3884-3891(1995)). Moreover, as shown in Table 2, the ligand specificity of thetwo responses also was similar, in that PLC activation by hPTH(1-34) andhPTHrP(1-36) was equivalent within each cell line and neither hPTH(3-34)nor hPTH(7-34) exhibited any consistently detectable activity in eithercase. Overall, these results did not suggest important differences inligand-receptor coupling or selectivity between the transient PLCresponse in HKRK B28 and the sustained response in HKRK B7.

Table 2. Activation of Phospholipase-C by PTH or PTHrP Peptides in HKRKB7 and HKRK B28 Cells.

HKRK B7 cells and HKRK B28 cells, previously loaded with[³H]myo-inositol, were incubated with each peptide (1000 nM) for 30 minor 4 min, respectively. The formation of IP₃ is expressed as apercentage of the amount measured in vehicle-treated controls. Valuesshown are means±SEM of triplicate determinations.

TABLE 2 Peptide HKRK B7 HKRK B28 (1000 nM) (30 min stimulation) (4 minstimulation) control 100 ± 5 100 ± 3 hPTH (1-34)  298 ± 24 172 ± 9 hPTH(1-31)  275 ± 21  170 ± 14 hPTH (3-34) 103 ± 1  85 ± 9 hPTH (7-34) 122 ±7  92 ± 10 hPTHrP (1-36)  93 ± 6 160 ± 9

Among the expected consequences of PLC activation via the human PTHR inLLC-PK1 cells would be release of sequestered calcium from intracellularstores (Bringhurst, F. R., el al., Endocrinology 132:2090-2098 (1993),Dunlay, R., and Hruska, K., Am J Physiol 258:F223-231(1990), Yamaguchi,D. T., et al., J Biol Chem 262:7711-7718 (1987), Pines, M., et al., Bone18:381-389 (1996)). Accordingly, cytosolic free calcium was measured inHKRK B28 and HKRK B7 cells following exposure to hPTH(1-34). As shown inFIG. 5, addition of hPTH(1-34) (100 nM) to monolayers of Fura 2AM-loadedcells caused a rapid elevation of cytosolic free calcium that peaked at400-600 nM within 20 sec and slowly decayed thereafter. No significantdifference in the magnitude or time-course of this response was observedbetween HKRK B7 and HKRK B28 (cf FIGS. 5A and 5C), despite the largerIP₃ response in the HKRK B7 cells (FIG. 4A).

To examine the possibility that activation of the cAMP pathway might beresponsible for the cytosolic free calcium response to PTH in thesecells, AB45 cells were studied, in which human PTHRs (370,000/cell) wereco-expressed with REV AB, a dominant-negative inhibitor of both basaland hormone-stimulated protein kinase A (PKA) (Clegg, C. H., et al., JBiol Chem 262:13111-13119 (1987), Fukayama, S., et al., Endocrinology134:1851-1858 (1994)). As shown in FIG. 5D, a robust cytosolic calciumresponse to hPTH(1-34) still was observed in these cAMP-resistant cellsand was of comparable magnitude to that observed in cAMP-responsive HKRKB28 cells that expressed a similar number of hPTHRs. Thus, the rapidincrease in cytosolic free calcium triggered by activation of the hPTHRin these cells apparently is not mediated by cAMP. Interestingly,however, it was observed that the calcium transient decayed more rapidlyin the cAMP-resistant AB45 cells than in HKRK B28 cells, approachingbasal levels within only 2-3 min (FIG. 5D). This occurred despite thefact that the magnitude and temporal pattern of IP₃ formation in AB45cells was nearly identical with that in HKRK B28 cells (data not shown).Neither 8BrcAMP (1 mM) nor the active phorbol ester TPA (100 nM) evokeda rapid calcium response (not shown). These results implied that themagnitude of the initial cytosolic free calcium response was mostclosely related to PLC activation, whereas the duration of the responsemay have been influenced predominantly by activation of the PKA pathway,possibly through enhanced entry of extracellular calcium (Yamaguchi, D.T., et al., J Biol Chem 262:7711-7718 (1987)).

Regulation of Biologic Responses by PTH

To determine if the changes in signaling efficiency and selectivityobserved at different levels of human PTHR expression in LLC-PK 1 cellscould result in alterations in integrated distal biologic responses toPTH, two well-characterized hormonal responses in these cells werestudied—secretion of u-PA and sodium-dependent active transport ofinorganic phosphate.

Secretion of urokinase-type plasminogen activator (u-PA) by LLC-PK1cells previously had been reported in response to activation of both thePKA and PKC pathways by calcitonin (Jans, D. A., and Hemmings, B. A.,FEBS Lett 205:127-131 (1986)). This assay was adapted to provide aconvenient spectrophotometric bioassay to report activation of AC or PLCvia the human PTHR in these cells. As shown in FIG. 6, incubation withhPTH(1-34) for 16 hr elicited a dose-dependent increase in u-PAsecretion from both HKRK B7 and HKRK B28 cells. In both cell lines,8BrcAMP and TPA, pharmacologic activators of PKA and PKC respectively,also strikingly induced this activity, reconfirming that both PKA andPKC pathways were linked to u-PA production. Maximal production of u-PAby HKRK B7 in response to hPTH(1-34) was greater than that in HKRK B28,both in absolute terms and in relation to other agonists such ascalcitonin or 8BrcAMP (FIG. 6).

Because the sensitivities and magnitudes of the cAMP responses to PTH inthese two cell lines were identical, it seemed possible that the greatermaximal u-PA response to PTH observed in HKRK B7 versus HKRK B28 cellsmight reflect the sustained activation of PLC seen only in the HKRK B7cells. Moreover, the EC₅₀ for stimulation of u-PA production byhPTH(1-34) (10-20 nM) was more similar to that for activation of IP₃formation (20-30 nM) than for cAMP accumulation (1 nM), particularly inHKRK B7 cells. Also, the maximal u-PA response to PTH was higher thanthat of 8BrcAMP in HKRK B7 (but not in HKRK B28 cells), consistent withactivation, by the more abundant hPTMs in these cells, of an additional,PKA-independent mechanism. Further evidence for involvement of acAMP-independent (presumably PKC-dependent) mechanism of PTH-inducedu-PA secretion in these cells was obtained using the cAMP-resistant AB45cells (FIG. 7). As expected, the u-PA response to 8BrcAMP was nearlyobliterated in these cells, whereas that to phorbol ester waswell-maintained. The PTH response, like that to calcitonin, wasdramatically attenuated but remained significantly greater than thatachieved with a maximal concentration of 8BrcAMP. This result furthersupports a role for mechanisms independent of PKA in mediatingPTH-stimulated u-PA secretion in these cells.

It was previously reported that stimulation of sodium-dependentphosphate uptake following activation of the rat PTHR in LLC-PK1 cellswas mediated principally by the PLC/PKC pathway (Guo, J., et al.,Endocrinology 136:3884-3891 (1995)) Similar observations have beenreported by others in CHO cells (Kaufmann, M., Mol Cell Endocrinol104:21-27 (1994)). The regulation of this response by PTH in HKRK B7 andHKRK B28 cells that expressed the human PTHR was therefore examined. Asshown in FIG. 8, phosphate uptake was stimulated significantly byhPTH(1-34) in these cells. This PKC-dependent action of PTH(1-34) wasobserved in both HKRK B28 and HKRK B7 cells, suggesting that thetransient PLC response observed in the HKRK B28 cells was sufficient toinduce this more sustained biologic response. This conclusion wasfurther supported by the observation that phosphate uptake also wasstimulated over 2-fold by PTH(1-34) in the cAMP-resistant AB45 cells(data not shown), in which, as noted above, the only measurable PTHRsignal was the transient stimulation of PLC and associated increase incytosolic free calcium.

Effects of changes in PTHR density upon ligand-dependent activation ofthe AC and PLC effectors were observed that, although obtained using anonhomologous model system in vitro, could have important physiologicimplications. Differences in specific patterns and relative intensitiesof the intracellular second messenger signals generated by PTHRactivation, particularly in osteoblasts or marrow stromal cells, may becritical in transmitting to both the immediate target cell andneighboring cells of the osteoclastic lineage information about varioustemporal patterns and concentration profiles of circulating PTH thatultimately may have profoundly different effects on bone (Dempster, D.W., et al., Endocrine Rev 14:690-709 (1993)). Accordingly, insights intopossible mechanisms of such differential signaling may be crucial forunderstanding the distinct mechanisms that must underlie the anabolicand catabolic effects of PTH on bone in vivo. The influence of receptorexpression on the “signaling phenotype” of the PTHR also could beimportant in modulating responses of chondrocytes to high localconcentrations of PTHrP in developing bone (Lanske, B., et al., Science273:663-666 (1996), Lee, K., et al., Endocrinology 137:5109-5118 (1996))

One mechanism for such differential signaling, based simply upon changesin the extracellular ligand concentration, is evident from previousstudies in which substantial differences in ligand sensitivity (EC₅₀) ofthe AC and PLC responses to PTH have been observed upon initial exposureof naive cells to PTH (Schneider, H., Eur J Pharmacol 246:149-155(1993), Pines, M., et al., Endocrinology 135:1713-1716 (1994), Guo, J.,et al., Endocrinology 136:3884-3891 (1995), Pines, M., et al., Bone18:381-389 (1996), Kaufmann, M., Mol Cell Endocrinol 104:21-27 (1994)).The present inventors report similarly striking differences insensitivity of the AC and PLC responses to PTH in LLC-PK1 cells thatexpress hPTHRs—i.e. the EC₅₀ that was observed for activation of AC was10-100-fold lower than that for PLC stimulation.

Another mechanism of differential signaling could involvedisproportionate desensitization of PTHR coupling to different Gproteins following prior exposure to ligand. Desensitization of PTHRsignaling has been widely described (Mitchell, J., and Goltzman, D.,Endocrinology 126:2650-2660 (1990), Abou-Samra, A. B., et al.,Endocrinology 135:2588-2594 (1994), Fukayama, S., et al., Endocrinology134:1851-1858 (1994), Yamamoto, I., et al., Endocrinology 122:1208-1217(1988), Freyaldenhoven, A. M., et al., Am J Physiol 262:E87-E95 (1992)),and differences noted among the various transduction pathways withrespect to the temporal patterns and magnitudes of these desensitizationevents also could modulate the relative intensity of subsequentsignaling along each route.

A third mechanism for generating differential signaling was suggested bythe previous finding that the relative magnitudes of the maximal AC andPLC signals were strikingly different, depending upon the number of ratPTHRs expressed on the cell surface (Guo, J., et al., Endocrinology136:3884-3891 (1995)). Thus, for any given extracellular ligandconcentration, variations in the number of cell-surface rPTHRsinfluenced not only the magnitude of a given intracellular signal, butalso its intensity relative to that of other signals, thereby alteringthe pattern of intracellular signal transduction independently ofchanges in ligand concentration. The present studies indicate that thisdifferential influence of receptor expression on AC versus PLC signalingefficiency may apply to human PTHRs as well and may occur without anychange in the EC₅₀'s of the individual responses. Analogous resultsrecently were reported for comparisons of AC and cytosolic free calciumsignaling in HEK-293 human kidney cells that expressed different numbersof human PTHRs (Pines, M., et al., Bone 18:381-389 (1996)). Thisdissociation of changes in ligand sensitivity from changes in efficiencyof G protein and/or effector coupling suggests that the structuraldeterminants of the PTHR involved in agonist recognition and activationmay operate relatively independently of those involved in Gprotein/effector interaction. The physiologic relevance of theseobservations is supported by the substantial changes in expression ofthe PTHR known to occur in cells of bone or during repeated PTH exposurein vitro or in vivo (Mitchell, J., and Goltzman, D., Endocrinology126:2650-2660 (1990), Abou-Samra, A. B., et al., Endocrinology135:2588-2594 (1994), Fukayama, S., et al., Endocrinology 134:1851-1858(1994), Yamamoto, I., et al., Endocrinology 122:1208-1217 (1988);Freyaldenhoven, A M., et al., Am J Physiol 262:E87-E95 (1992); Forte,L., et al., Am J Physiol 242:E 154-E163 (1982); Bellorin, F. E., et al.,Kidney Int 47:38-44 (1995)). Moreover, variation of endogenous PTHRexpression within the range that was studied in LLC-PK1 cells has beendescribed in other cell systems in which endogenous PTHR genes areexpressed (Yamamoto, I., et al., Endocrinology 122:1208-1217 (1988),Shukunami, C., et al., J Cell Biol 133:457-468 (1996)).

Several additional aspects of the signaling properties of hPTHRs thatwere have observed here are worthy of comment. First, it is of interestthat the efficiencies with which the human PTHR couples to the AC andPLC responses in LLC-PK1 cells were substantially and coordinatelyreduced when compared with those for the corresponding responses incells expressing rat PTHRs. This is very evident in FIG. 3, for example,where roughly 3-5-fold more human than rat PTHRs were required toachieve comparable maximal activation of PLC. An analogous difference inefficiency of AC coupling is suggested by comparing the data for thehPTHR in Table 1 with results that were previously reported for ratPTHRs in these cells (Guo, J., et al., Endocrinology 136:3884-3891(1995)). This “rightward shift” of the relation between overallsignaling efficiency and receptor density may reflect an intrinsicproperty of the human PTHR or, alternatively, a relative speciesincompatibility between the human receptor and the intracellular signaltransducers expressed by the porcine cells used here. More efficientcoupling of transfected human PTHRs to PLC or cytosolic calciumsignaling has been observed in cells of primate or human origin (Pines,M., et al., Bone 18:381-389(1996), Schipani, E., et al., Science268:98-100 (1995)). Direct comparisons of the functions of transfectedrat and human PTHRs in cells derived from a wider range of species willbe needed to clarify this issue.

Second, it was found that not only the magnitude but also the temporalprofile of the IP₃ response (at maximal concentrations of ligand) wasrelated to receptor density. Specifically, the PLC response at lowerlevels of PTHR expression was transient, lasting less than 10 min,whereas that triggered at higher PTHR densities was considerably moresustained. The explanation for this difference is not immediately clear.The mechanism(s) of the rapid decline from the peak of measured IP₃ thatwas observed in the HKRK B28 cells, which is typical of many receptorresponses (Berridge, M., and Irvine, R., Nature 341:197-205 (1989)),probably is complex and could include (a) termination of the initialstimulus (via receptor desensitization or internalization), (b)depletion of a limited pool of membrane phosphatidylinostitol4,5-bisphosphate substrate accessible to the PTHR (especially in thepresence of lithium (Nahorski, S. R., et al., Trends Pharmacol Sci12:297-303 (1991))), or (c) rapid induction of enzymes that metabolizeinositol polyphosphates—notably the inositol 1,4,5-trisphosphate5-phosphatase—which may be mediated in part by activation of PKC(Nahorski, S. R, et al., Trends Pharmacol Sci 12:297-303 (1991), Shears,S. B., Biochem J 260:313-324 (1989)). Substrate depletion seems anunlikely explanation in the system, as the peak rate of substratehydrolysis and IP₃ formation at 4 min actually was greater in the HKRKB7 cells with the more prolonged response. It is not yet known, however,if the distribution of expressed PTHRs is uniform throughout themembranes of these polarized epithelial cells at all levels of hPTHRexpression that were studied. It is possible, for example, that PTHRdistribution might be more restricted at lower rates of receptorsynthesis, which could limit the size of the substrate pool available tothe receptor in such cells and permit “local” substrate depletion. Itwas considered that the more prolonged PLC response in HKRK B7 cellscould be due simply to a more intense and protracted activation of theenzyme, sustained by a much larger number of occupied receptors held inan active configuration in these cells, that was sufficient to nearlysaturate the phosphatases involved in metabolizing IP₃ (Shears, S. B.,Biochem J 260:313-324 (1989)). The results seem most consistent withthis possibility, as no difference could be detected in EC₅₀'s of theresponses measured at 4 min vs. 30 min, nor was an obvious differencedetected in ligand selectivity that might have pointed to two differentmechanisms. In contrast to these differences in the PLC responses,activation of AC by PTH appeared to proceed identically over the first15 min in both cell lines. Moreover, in experiments not shown, it wasalso verified, by direct measurement of enzyme activity at intervals upto 24 hr after addition of 1000 nM hPTH(1-34), that this is true of themore sustained PKA responses in these cells as well.

A third interesting feature of hPTHR signaling in these cells concernsthe cytosolic calcium responses that were observed. Given thedifferences discussed above in PLC activation profiles between HKRK B28and HKRK B7 cells, the widely accepted view that IP₃ is a majordeterminant of calcium release from intracellular stores duringactivation of G protein-coupled receptors (Berridge, M. J., Nature361:315-325 (1993)), and the recent demonstration, in permeabilizedHEK-293 cells expressing human PTHRs, that PTH and IP₃ appear to competefor release from the same intracellular pool of calcium (Pines, M., etal., Bone 18:381-389 (1996)), it is perhaps surprising that thecytosolic free calcium transients triggered by PTH in these two celllines were virtually identical, both in time and magnitude. The resultswith cAMP-resistant AB45 cells did suggest that the sustained phase ofcytosolic free calcium elevation may be related to activation ofPKA—possibly by promoting entry of extracellular calcium (Yamaguchi, D.T., et al., J Biol Chem 262:7711-7718 (1987))—but the initial phase ofthe calcium response appeared to be independent of PKA. Further, otherexperiments, in which forskolin, 8-bromo-cAMP and phorbol ester alsofailed to elicit calcium responses, offered no evidence for directinvolvement of PKA or PKC in eliciting this response. It was concludedthat the rapid cytosolic calcium response to PTH is most closely relatedto the initial increase in IP₃ in these cells and, further, that theamount of IP₃ generated acutely in HKRK B28 cells, although less thanthat produced in HKRK B7 cells, must be sufficient to achieve maximalrelease of calcium from the IP₃-sensitive intracellular pool.

Signaling and Biological Activity of hPTH Analogs

Several PTH analogs were reported to exhibit signal-selectivity inPTH-responsive cells of non-human origin. These include PTH(3-34) andPTH(7-34), which have been shown in some systems to selectively activatePKC and/or cytosolic calcium transients (Fujimori, A., et al.,Endocrinology 128:3032-3039 (1991), Abou-Samra, A. B., et al.,Endocrinology 135:2588-2594 (1994); Azarani, A., et al., J Biol Chem271:14931-14936 (1996); Chakravarthy, B. R, et al., Biochem Biophys ResCommun 171:1105-1110 (1990); Fujimori, A., et al., Endocrinology130:29-36 (1992); Jouishomme, H., et al., Endocrinology 130:53-60(1992); Janulis, M., et al., Endocrinology 133:713-719 (1993)), andPTH(1-31), which has been reported to activate AC but not PKC in ratosteosarcoma cells and primary rat spleen cells (Rixon, R. H., et al., JBone Miner Res 9:1179-1189 (1994), Whitfield, J. F., and Morley, P.,Trends Pharmacol Sci 16:382-386 (1995), Jouishomme, H., et al.,Endocrinology 130:53-60 (1992)). Because such fragments have beenconsidered as candidates for clinical use (Dempster, D. W., et al.,Endocrine Rev 14:690-709 (1993), Whitfield, J. F., and Morley, P.,Trends Pharmacol Sci 16:382-386 (1995)) it was determined if theseanalogs would display the expected signal-selectivity via human PTHRs.

As shown in FIG. 2B, activation of AC in HKRK B28 cells was stimulatedsignificantly by hPTH(3-34) at concentrations of 10 nM and above,although the response to 1000 nM of this peptide was only 16% of themaximal response to hPTH(1-34). Human PTH(7-34) failed to activate AC atall concentrations tested, whereas the responses to hPTHrP(1-36) andhPTH(1-31) were indistinguishable from that to hPTH(1-34). Similarresults were obtained in experiments with HKRK B7 cells (data notshown).

When tested at concentrations of 1000 nM, neither hPTH(3-34) norhPTH(7-34) activated PLC significantly, even in HKRK B7 cells thatexpressed nearly 1 million receptors per cell (Table 2). In contrast,both hPTHrP (1-36) and, unexpectedly, hPTH(1-31) stimulated PLCequivalently to hPTH(1-34) in both HKRK B28 (at 4 min) and HKRK B7 (at30 min). Human PTH(1-31) (100 nM) also fully activated cytosolic freecalcium transients in HKRK B7 cells (FIG. 5B), whereas neitherhPTH(3-34) nor hPTH(7-34) (100 nM) induced this response (not shown).

In the u-PA bioassay, which is mainly dependent upon PKA activation butalso reflects PKA-independent signaling (as described above), theactivities of hPTH(3-34), hPTH(7-34) and hPTH(1-31) were concordant withtheir signaling properties. Thus, hPTH(3-34) was partially active(50-60% of maximal response to hPTH(1-34)), whereas hPTH(7-34) wasinactive and hPTH(1-31) was fully active (Table 3, FIG. 9). Notably,maximal activation of u-PA secretion by hPTH(1-31) was identical to thatof hPTH(1-34) even in HKRK B7 cells (FIG. 9, Table 3), where maximal PTHactivity exceeds that achieved with maximal pharmacologic activation ofPKA with 8BrcAMP, and in AB45 cells (FIG. 7), where PKA (and the actionof 8BrcAMP) is almost completely blocked and the residual activity ofPTH is presumably mediated almost entirely by PKA-independent signals.Similarly, in both HKRK B28 and HKRK B7 cells, hPTH(1-31) and hPTH(1-34)(as well as hPTHrP(1-36)) showed comparable maximal activation ofphosphate uptake, which is PKC-dependent in these cells (Guo, J., etal., Endocrinology 136:3884-3891 (1995)), whereas hPTH(3-34) andhPTH(7-34) were inactive, as expected (FIG. 8).

Table 3. Stimulation of u-PA Secretion by hPTH/PTHrp Peptides in HKRK B7and HKRK B28 Cells

Cells were incubated with the indicated peptides (1000 nM) for 16 hrbefore measurement of secreted u-PA present in the medium, which wasexpressed as a percentage of the activity measured in vehicle-treatedcontrols. Values shown are means±SEM of triplicate determinations.

TABLE 3 Peptide (1000 nM) HKRK B7 HKRK B28 hPTH (1-34) 100 ± 5 100 ± 8hPTH (1-31) 101 ± 9 101 ± 6 hPTH (3-34)  57 ± 3  50 ± 1 hPTH (7-34)  1 ±1  0 ± 2 hPTHrP (1-36)  88 ± 5  97 ± 9

The full equivalence of the signaling and biologic properties ofhPTH(1-31) and hPTH(1-34) that was observed via transfected human PTHRsin these LLC-PK1 cells was quite unexpected, as this analog has beenreported to be devoid of activity in cellular bioassays of PKCactivation, despite full potency with respect to stimulation of AC, inrat systems (Rixon, R. H., et al., J Bone Miner Res 9:1179-1189 (1994),Jouishomme, H., et al., Endocrinology 130:53-60 (1992), Neugebauer, W.,et al., Biochemistry 34:8835-8842 (1995)). It was determined if theapparent discrepancy between the findings of the present inventors andthe previous reports might relate to the difference in receptor species.Accordingly, the effects of hPTH(1-34) and hPTH(1-31) in LLC-PK1 cells(EW5) that express rat PTHRs were compared. Both peptides activated ACequivalently in EW 5 cells (FIG. 10). As shown in Table 4, maximal IP₃generation by hPTH(1-31) via the rat PTHR in EW5 cells (and in 3 otherrPTHR-expressing cell lines) was approximately 70% as great as that byhPTH(1-34), whereas u-PA production at 16 hr was identical in responseto 100 nM of either peptide. Stimulation of phosphate uptake by the twopeptides also was identical. Thus, the ability of hPTH(1-31) to activatePLC and other cAMP-independent responses via the PTHR was not restrictedto the human receptor but was seen also in LLC-PK1 cells that expressrecombinant rat PTHRs. The PLC response to hPTH(1-31) may be modestlyimpaired, relative to hPTH(1-34), via the rat PTHR, whereas the humanreceptor seems unable to discriminate between these two ligands.

Table 4. Comparison of hPTH(1-34) and hPTH(1-31) in LLC-PK1 CellsExpressing Rat PTHR.

Responses to hPTH(1-34) or hPTH(1-31) (1000 nM) were measured in EW5cells, which express 320,000 rat PTHRs per cell. Each value is themean±SEM of a representative experiment performed in triplicate. Similarresults were obtained in at least 3 individual experiments. The modestreduction in IP₃ formation observed with hPTH(1-31) relative tohPTH(1-34) was confirmed in 3 other LLC-PK1 cell lines that expressedrat PTHRs (i.e. EW29, AR-C38 and AR-B44, which express 190,000, 630,000and 800,000 PTHRs/cell, respectively), in which maximal IP₃ formationafter hPTH(1-31) (as % of hPTH(1-34) effect) was 75%, 77% and 64%,respectively.

TABLE 4 Peptide IP₃ Formation Phosphate uptake u-PA Activity (1000 nM)(% of basal) (% of basal) (units/well) control 100 ± 5  100 ± 2  0.8 ±0.3 hPTH (1-34) 444 ± 31 130 ± 2 43.5 ± 1.8 hPTH (1-31) 316 ± 35 134 ± 341.2 ± 1.2

In view of the widely postulated linkage(s) between specificPTHR-generated second messenger signals and certain biologic actions ofthe hormone in tissues such as bone or kidney (Dempster, D. W., et al.,Endocrine Rev 14:690-709 (1993), Rixon, R. H., et al., J. Bone Miner Res9:1179-1189 (1994), Whitfield, J. F., and Morley, P., Trends PharmacolSci 16:382-386 (1995), Dunlay, R., and Hruska, K., Am J. Physiol258:F223-231 (1990), Janulis, M., et al., Endocrinology 133:713-719(1993)), it was of great interest to examine the properties of severaltruncated hPTH(1-34) analogs in cells expressing the human PTHR.

Weak but significant activation of AC (and u-PA secretion) by hPTH(3-34)but no PLC- or cytosolic calcium-stimulating activity was observed.These results contrast with other reports, based mainly in rodentmodels, in which PTH(3-34) peptides generally have exhibited loss of ACactivation with relative preservation of PLC/PKC- or calcium-stimulatingactivity (Fujimori, A., et al., Endocrinology 128:3032-3039 (1991),Abou-Samra, A. B., et al., Endocrinology 135:2588-2594 (1994); AzaraniA, et al., J Biol Chem 271:14931-14936 (1996); Chakravarthy, B. R., etal., Biochem Biophys Res Commun 171:1105-1110 (1990), Fujimori, A., etal., Endocrinology 130:29-36 (1992); Jouishomme, H., et al.,Endocrinology 130:53-60 (1992); Janulis, M., et al., Endocrinology133:713-719 (1993), Azarani, A., et al., J Biol Chem 270:23166-23172(1995)). In transfected HEK 293 cells that expressed 400,000 human PTHRsper cell, Pines et al. observed slight but significant increases in bothAC and cytosolic free calcium in response to 10 nM [Nle^(8,18),Tyr³⁴]bPTH(3-34)amide (Pines, M., et al., Bone 18:381-389 (1996)),whereas no PLC or calcium responses to hPTH(3-34)amide at 100 nM wasobserved. On the other hand, others also have failed to detectactivation of PKC or cytosolic calcium transients by PTH(3-34) peptidesin rat and other non-human cell systems (Reid, I., et al., Am J Physiol252:E45-E51 (1987); Civitelli, R., et al., Endocrinology 125:1204-1210(1989); Tamura, T., et al., Biochem Biophys Res Commun 159:1352-1358(1989)). It thus seems likely that these disparities may be attributableto differences in experimental systems or in the species and structureof the PTHRs involved or of the particular PTH(3-34) peptides employed.

More surprising was the finding that hPTH(1-31) was fully equivalent tohPTH(1-34) with respect to activation of AC, PLC, cytosolic calciumtransients, u-PA secretion and phosphate uptake in LLC-PK1 cells thatexpress hPTHRs. Previously, hPTH(1-31) had been reported to retain ACactivity but to be devoid of PKC-stimulating activity (Rixon, R. H., etal., J Bone Miner Res 9:1179-1189 (1994), Jouishomme, H., et al., J BoneMiner Res 9:943-949 (1994), Neugebauer, W., et al., Biochemistry34:8835-8842(1995)). These observations formed the basis of the proposalthat the region hPTH(Jouishomme, H., et al., J Bone Miner Res 9:943-949(1994), Schipani, E., et al., Science 268:98-100 (1995); Clegg, C. H.,et al., J Biol Chem 262: 13111-13119 (1987); Menniti, F., et al., MolPharmacol 40:727-733 (1991); Fukayama, S., et al., Endocrinology134:1851-1858 (1994)) comprises a core domain for activation of PKC viathe PTHR (Whitfield, J. F., and Morley, P., Trends Pharmacol Sci16:382-386 (1995), Jouishomme, H., et al., J Bone Miner Res 9:943-949(1994)). Thus, experiments in which ovariectomized rats given dailyinjections of hPTH(1-31) exhibited increases in bone mass comparable tothose seen with hPTH(1-34) have been widely interpreted as evidence of aprimary role for the cAMP/PKA cascade in the anabolic effect of PTH onbone (Rixon, R. H., et al., J Bone Miner Res 9:1179-1189 (1994),Whitfield, J. F., et al., Calcif Tissue Int 58:81-87 (1996)). AlthoughPKC was not measured directly, both a cytosolic calcium response tohPTH(1-31) that could not be mimicked by cAMP analogs and, as well,vigorous stimulation of phosphate uptake, a biologic response shownpreviously to be linked primarily to activation of PKC in these cellswas observed(Guo, J., et al., Endocrinology 136:3884-3891 (1995)). Thus,the direct demonstration of PLC activation by hPTH(1-31) via the hPTHRis notable and raises the important possibility that the ligandselectivity of the human PTHR may differ substantially from that of therat PTHR (given that all previous work had been conducted in ratsystems). Such a conclusion would have important implications forefforts aimed at developing clinically useful signal-selective PTHanalogs on the basis of bioassays in rats (Whitfield, J. F., and Morley,P., Trends Pharmacol Sci 16:382-386 (1995)).

To address this issue further, the activities of hPTH(1-34) andhPTH(1-31) in LLC-PK1 cells that expressed rat PTHRs were compared.Surprisingly, it was found that the activities of the two peptides arenearly indistinguishable in this system as well, although maximal PLCactivation by hPTH(1-31) was reduced significantly—to 60% of the maximalresponse to hPTH(1-34). These findings are inconsistent with previouscharacterizations of hPTH(1-31) as a cAMP-selective analog in rat cellsand indicate that the full range of cAMP-independent activities ofhPTH(1-31) that were observed via hPTHRs in LLC-PK1 cells are notentirely unique to the human PTHR. Therefore, the difference in PTHRspecies per se would appear not to explain the disparity between thepresent findings in the HKRK cells and those previously reported byothers using rat cells (Rixon, R. H., et al., J Bone Miner Res9:1179-1189 (1994), Jouishomme, H., et al., J Bone Miner Res 9:943-949(1994), Neugebauer, W., et al., Biochemistry 34:8835-8842 (1995)).

There are several possible explanations for the apparent differencesbetween the present findings and those from previous investigations ofthe hPTH(1-31) peptide. First, different responses have been measured.PLC, cytosolic free calcium and certain distal biologic effects known tobe linked to PLC/PKC activation in LLC-PK1 cells were measured, whereasJouishomme et al. measured PKC in unextracted membranes but not PLC orcytosolic free calcium (Rixon, R. H., et al., J Bone Miner Res9:1179-1189 (1994), Jouishomme, H., et al., J Bone Miner Res9:943-949(1994)). It is known that PKC may be regulated by upstreameffectors other than PLC (Exton, J., J Biol Chem 265:1-4 (1990)).Second, previous work was conducted using ROS 17/2 osteosarcoma cells orprimary cultures of rat splenic lymphocytes that express endogenous,rather than transfected, rat PTH receptor genes (Rixon, R. H., et al., JBone Miner Res 9:1179-1189 (1994), Jouishomme, H., et al., J Bone MinerRes 9:943-949 (1994)). The ROS 17/2 cells almost certainly express thesame rPTHR that were studied here, as they were the source of the ROS17/2.8 subclone from which the rPTHR cDNA originally was cloned and thenused to create the rPTHR-expressing LLC-PK1 cells used in theexperiments (Abou-Samra, A. B., et al., Proc Natl Acad Sci USA89:2732-2736 (1992), Bringhurst, F. R., et al., Endocrinology132:2090-2098 (1993), Guo, J., et al., Endocrinology 136:3884-3891(1995)). On the other hand, it is quite possible that ROS 17/2 cells andspleen cells express alternate species of receptors for PTH that couldhave influenced the response(s) to hPTH(1-31) in these cells. Forexample, a PTH receptor with apparent C-terminal specificity, not yetfully characterized, has been described in ROS 17/2.8 cells (Inomata,N., et al., Endocrinology 136:4732-4740). Third, the ROS 17/2 and ratspleen cell systems differ in other ways from the rPTHR-transfectedLLC-PK1 cells that were studied: (a) the surface expression of PTHRs isknown, or likely, to be lower than that in the LLC-PK1 cells were used,(b) rPTHRs are coupled to rat rather than porcine G proteins, and (c)the relative quantities of the various subtypes of expressed G proteinsand effector enzymes could be very different. The issue of lower PTHRexpression may be particularly important. The data suggest that thepotency of the hPTH(1-31) peptide for PLC/PKC activation via the ratPTHR is moderately reduced (by 40%), relative to that of hPTH(1-34).Given the important influence of receptor density upon PLC signalingthat was described, it seems possible that this difference in potencyfor PLC between hPTH(1-34) and hPTH(1-31) could be amplified in cellsthat express fewer rPTHRs than do the LLC-PK1 cells used here. On theother hand, no loss of PLC-stimulating potency via the human PTHR forhPTH(1-31) was found, even in cells (HKRK B28) in which human PTHRsurface density was close to the minimum required to detect this signal.

All references mentioned herein are incorporated by reference in thedisclosure. Having now fully described the invention by way ofillustration and example for purposes of clarity and understanding, itwill be apparent to those of ordinary skill in the art that certainchanges and modifications may be made in the disclosed embodiments andsuch modifications are intended to be within the scope of the presentinvention. As examples, the preferred embodiments constitute only oneform of carrying out the claimed invention.

1. A method for determining whether a compound of interest is an agonistof a receptor which couples to both Gs and Gq proteins thereby affectingan adenylyl cyclase or phospholipase C pathway said method comprising:(a) introducing into a first and second cell line, an expression vectorcomprising a nucleotide sequence encoding said receptor which is notnormally expressed in said first and second cell lines, wherein saidfirst and second cell lines express urokinase-type plasminogen activator(u-PA) and said second cell line also has inhibited Gs signaling of u-PAactivity relative to said first cell line; (b) contacting said first andsecond cell lines with said compound of interest; (c) measuring the u-PAactivity by fluorescence or absorbance spectroscopy of the cell culturesupernatant of step (b) and the cell culture supernatant of said firstand second cell lines which have not been in contact with said compoundof interest; and (d) determining whether said compound of interest is anagonist of a receptor which couples to both Gs and Gq proteins therebyaffecting an adenylyl cyclase or phospholipase C pathway, wherein theu-PA activity of the supernatant from said first and second cell linesfrom step (b) is greater than the u-PA activity of the supernatant fromsaid first and second cell lines which have not been in contact withsaid compound of interest.
 2. The method of claim 1, wherein said Gs andGq protein coupled receptor is human PTHR.
 3. The method of claim 1,wherein said first cell line and said second cell line is LLC-PK1.
 4. Amethod for determining whether a compound of interest is an agonist of areceptor which couples to both Gs and Gq proteins thereby affecting anadenylyl cyclase or phospholipase C pathway said method comprising: (a)providing a first and second cell line, wherein said first and secondcell lines express urokinase-type plasminogen activator (u-PA) andwherein said second cell line also has inhibited Gs signaling of u-PAactivity relative to said first cell line; (b) introducing into a groupof said first and second cell lines, an expression vector comprising anucleotide sequence encoding said receptor which is not normallyexpressed in said first and second cell lines, thereby providing stablytransfected cells of said first and second cell lines; (c) contactingsaid stably transfected cells of step (b) and said first and second celllines of step (a), which are not stably transfected with said receptor,with said compound; (d) measuring the u-PA activity, by fluorescence orabsorbance spectroscopy, of the cell culture supernatant of said stablytransfected cells of step (c) and the cell culture supernatant of saidfirst and second cell lines of step (c); and (e) determining whethersaid compound of interest is an agonist of a receptor which couples toboth Gs and Gq proteins thereby affecting an adenylyl cyclase orphospholipase C pathway, wherein the u-PA activity of the supernatantfrom said stably transfected cells of step (c) is greater than the u-PAactivity of the supernatant of said first and second cell lines of step(c), which are not stably transfected with said receptor.
 5. The methodof claim 4, wherein said Gs and Gq protein coupled receptor is humanPTHR.
 6. The method of claim 4, wherein said first cell line and saidsecond cell line is LLC-PK1.
 7. A method for determining whether acompound of interest is an antagonist of a receptor which couples toboth Gs and Gq proteins thereby affecting an adenylyl cyclase orphospholipase C pathway said method comprising: (a) introducing into afirst and second cell line an expression vector comprising a nucleotidesequence encoding said receptor which is not normally expressed in saidfirst and second cell lines, wherein said first and second cell linesexpress urokinase-type plasminogen activator (u-PA) and said second cellline also has inhibited Gs signaling of u-PA activity relative to saidfirst cell line; (b) contacting a first group of said first and secondcell lines of step (a) with a known agonist of said receptor; (c)contacting a second group of said first and second cell lines of step(a) with a known agonist of said receptor and said compound of interest;(d) measuring the u-PA activity, by fluorescence or absorbancespectroscopy, of the cell culture supernatant of said first and secondgroups of steps (b) and (c); and (e) determining whether said compoundof interest is an antagonist of a receptor which couples to both Gs andGq proteins thereby affecting an adenylyl cyclase or phospholipase Cpathway, wherein the u-PA activity of the supernatant from said firstgroup of step (b) is greater than the u-PA activity of the supernatantof said second group of step (c).
 8. The method of claim 7, wherein saidGs and Gq protein coupled receptor is human PTHR.
 9. The method of claim7, wherein said first cell line and said second cell line is LLC-PK1.10. The method of claim 7, wherein said compound of interest isadministered after said agonist in step (c).
 11. The method of claim 7,wherein said compound of interest is administered before said agonist instep (c).
 12. The method of claim 7, wherein said compound of interestis administered concurrently as said agonist in step (c).